A cautionary note on the potential pitfalls of using N-terminal truncated CD63 to label small extracellular vesicles
Abstract Small extracellular vesicles (sEV) are nanosized vesicles that facilitate intracellular communication. A significant research obstacle is the isolation of sEV devoid of non-sEV contaminants. Immunoaffinity capture with sEV-specific antibodies is an attractive approach to purifying sEV, but...
Saved in:
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-03-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-025-91597-6 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849767421193224192 |
|---|---|
| author | Elias Sulaiman Derek M. Yellon Sean M. Davidson |
| author_facet | Elias Sulaiman Derek M. Yellon Sean M. Davidson |
| author_sort | Elias Sulaiman |
| collection | DOAJ |
| description | Abstract Small extracellular vesicles (sEV) are nanosized vesicles that facilitate intracellular communication. A significant research obstacle is the isolation of sEV devoid of non-sEV contaminants. Immunoaffinity capture with sEV-specific antibodies is an attractive approach to purifying sEV, but it risks disrupting the vesicles during antibody dissociation. Furthermore, immunoaffinity capture may require the modification of EV-specific proteins for the incorporation of tags on the EV surface, with unknown implications on EV production and function. The aim of this study was to investigate whether a previously reported CD63 truncation is efficient for the incorporation of small tags on the extravesicular surface. We therefore conjugated ALFA-tag to N-terminal-truncated CD63, and included nanoluciferase at the C-terminus, for luminescent tracing of the sEV. Full-length CD63-nanoluciferase was used as a control. Plasmid constructs expressing these proteins were transfected into HEK293 cells. In contrast to a previous report, the N-terminal truncation of CD63 impaired its membrane localisation and reduced the yield of EVs. Further investigation revealed that some of the tagged CD63 was co-localized with aggresomes and was preferentially secreted from the cells as soluble protein rather than being associated with sEV. These results demonstrate that CD63 truncation can impair its function and EV yield, potentially generating misleading results. |
| format | Article |
| id | doaj-art-870988bad4044fdfb9a2c7da0ffbc780 |
| institution | DOAJ |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-870988bad4044fdfb9a2c7da0ffbc7802025-08-20T03:04:12ZengNature PortfolioScientific Reports2045-23222025-03-011511910.1038/s41598-025-91597-6A cautionary note on the potential pitfalls of using N-terminal truncated CD63 to label small extracellular vesiclesElias Sulaiman0Derek M. Yellon1Sean M. Davidson2The Hatter Cardiovascular Institute, University College LondonThe Hatter Cardiovascular Institute, University College LondonThe Hatter Cardiovascular Institute, University College LondonAbstract Small extracellular vesicles (sEV) are nanosized vesicles that facilitate intracellular communication. A significant research obstacle is the isolation of sEV devoid of non-sEV contaminants. Immunoaffinity capture with sEV-specific antibodies is an attractive approach to purifying sEV, but it risks disrupting the vesicles during antibody dissociation. Furthermore, immunoaffinity capture may require the modification of EV-specific proteins for the incorporation of tags on the EV surface, with unknown implications on EV production and function. The aim of this study was to investigate whether a previously reported CD63 truncation is efficient for the incorporation of small tags on the extravesicular surface. We therefore conjugated ALFA-tag to N-terminal-truncated CD63, and included nanoluciferase at the C-terminus, for luminescent tracing of the sEV. Full-length CD63-nanoluciferase was used as a control. Plasmid constructs expressing these proteins were transfected into HEK293 cells. In contrast to a previous report, the N-terminal truncation of CD63 impaired its membrane localisation and reduced the yield of EVs. Further investigation revealed that some of the tagged CD63 was co-localized with aggresomes and was preferentially secreted from the cells as soluble protein rather than being associated with sEV. These results demonstrate that CD63 truncation can impair its function and EV yield, potentially generating misleading results.https://doi.org/10.1038/s41598-025-91597-6 |
| spellingShingle | Elias Sulaiman Derek M. Yellon Sean M. Davidson A cautionary note on the potential pitfalls of using N-terminal truncated CD63 to label small extracellular vesicles Scientific Reports |
| title | A cautionary note on the potential pitfalls of using N-terminal truncated CD63 to label small extracellular vesicles |
| title_full | A cautionary note on the potential pitfalls of using N-terminal truncated CD63 to label small extracellular vesicles |
| title_fullStr | A cautionary note on the potential pitfalls of using N-terminal truncated CD63 to label small extracellular vesicles |
| title_full_unstemmed | A cautionary note on the potential pitfalls of using N-terminal truncated CD63 to label small extracellular vesicles |
| title_short | A cautionary note on the potential pitfalls of using N-terminal truncated CD63 to label small extracellular vesicles |
| title_sort | cautionary note on the potential pitfalls of using n terminal truncated cd63 to label small extracellular vesicles |
| url | https://doi.org/10.1038/s41598-025-91597-6 |
| work_keys_str_mv | AT eliassulaiman acautionarynoteonthepotentialpitfallsofusingnterminaltruncatedcd63tolabelsmallextracellularvesicles AT derekmyellon acautionarynoteonthepotentialpitfallsofusingnterminaltruncatedcd63tolabelsmallextracellularvesicles AT seanmdavidson acautionarynoteonthepotentialpitfallsofusingnterminaltruncatedcd63tolabelsmallextracellularvesicles AT eliassulaiman cautionarynoteonthepotentialpitfallsofusingnterminaltruncatedcd63tolabelsmallextracellularvesicles AT derekmyellon cautionarynoteonthepotentialpitfallsofusingnterminaltruncatedcd63tolabelsmallextracellularvesicles AT seanmdavidson cautionarynoteonthepotentialpitfallsofusingnterminaltruncatedcd63tolabelsmallextracellularvesicles |